Serine-arginine-rich protein kinase-1 inhibition for the treatment of diabetic retinopathy.

Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.

Transmission Electron Microscopy of Endothelium.

Transmission electron microscopy using resin sections still remains an exceptionally useful tool in evaluating cellular ultrastucture within tissue. For the endothelium the best method for maintaining such structure is perfusion fixation: fixing the tissue under physiological pressure. Here the focus is on a method of maintaining the vascular wall structure including the endothelial glycocalyx and extending this with tilt series tomography. Shown are typical histological sections from multiple capillary beds including brain, heart and retina using a lanthanide staining technique (LaDy GAGa) to highlight that the differences in normo-physiology are substantial.It is hoped that users will find the notes useful in deciding which specific staining and imaging method would suit their needs so this technically challenging, and low throughput methodology, is used to its best effect.

Noninvasive Measurement of Retinal Microvascular Permeability During Loss of Endothelial Quiescence.

In the retina EC dysfunction and angiogenesis are driven by an altered microenvironment e.g., diabetes, leading to hypoxia and inflammation in the retinal layers, resulting in excessive vascular leakage and growth. The gold standard for measuring blood-retinal barrier permeability in response to disease and or therapy has been the gold standard Evans blue (EB) assay. However, this technique has limitations in vivo, including nonspecific tissue binding and toxicity. Here we describe a novel imaging methodology combining sodium fluorescein fundus angiography (FFA) with mathematical quantification allowing retinal permeability to be noninvasively and accurately measured at multiple time points in the same animal, minimizing animal use in line with the 3Rs framework. In addition, this technique is a nontoxic, high throughput, sensitive, and cost-effective alternative technique to the Evans blue assay. Moreover, this technique can be translated to other species.

Quantification of Angiogenesis in Laser Choroidal Neovascularization.

Murine laser-induced laser choroidal neovascularization is a widely used and robust model of wet (exudative) age-related macular degeneration (wAMD). wAMD is one of the leading causes of blindness in the Western world. In brief, a focused laser beam is used to penetrate Bruch's membrane, which separates the choriocapillaris (well-vascularized choroid layer) from the pigmented layers of the retina. Damage to the integrity of this membrane during diabetes leads to fluid accumulation and vascular invasion into the subretinal layers resulting in a progressive worsening of vision. Here we describe a 14-day model using untreated C57/Bl6 mice, but it is equally applicable to incorporation into transgenic studies and therapeutic agent development (such as eye drops), injection of therapeutic agents (including antibodies), and for longer time course studies. In vivo functional analysis or lesioned choroids can be studied with further immunohistochemical staining for further analyses.

Hydrogen Sulfide Is a Novel Protector of the Retinal Glycocalyx and Endothelial Permeability Barrier.

Significantly reduced levels of the anti-inflammatory gaseous transmitter hydrogen sulfide (H2S) are observed in diabetic patients and correlate with microvascular dysfunction. H2S may protect the microvasculature by preventing loss of the endothelial glycocalyx. We tested the hypothesis that H2S could prevent or treat retinal microvascular endothelial dysfunction in diabetes. Bovine retinal endothelial cells (BRECs) were exposed to normal (NG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) ± the slow-release H2S donor NaGYY4137 in vitro. Glycocalyx coverage (stained with WGA-FITC) and calcein-labeled monocyte adherence were measured. In vivo, fundus fluorescein angiography (FFA) was performed in normal and streptozotocin-induced (STZ) diabetic rats. Animals received intraocular injection of NaGYY4137 (1 µM) or the mitochondrial-targeted H2S donor AP39 (100 nM) simultaneously with STZ (prevention) or on day 6 after STZ (treatment), and the ratio of interstitial to vascular fluorescence was used to estimate apparent permeability. NaGYY4137 prevented HG-induced loss of BREC glycocalyx, increased monocyte binding to BRECs (p ≤ 0.001), and increased overall glycocalyx coverage (p ≤ 0.001). In rats, the STZ-induced increase in apparent retinal vascular permeability (p ≤ 0.01) was significantly prevented by pre-treatment with NaGYY4137 and AP39 (p < 0.05) and stabilized by their post-STZ administration. NaGYY4137 also reduced the number of acellular capillaries (collagen IV + /IB4-) in the diabetic retina in both groups (p ≤ 0.05). We conclude that NaGYY4137 and AP39 protected the retinal glycocalyx and endothelial permeability barrier from diabetes-associated loss of integrity and reduced the progression of diabetic retinopathy (DR). Hydrogen sulfide donors that target the glycocalyx may therefore be a therapeutic candidate for DR.

Non-invasive measurement of retinal permeability in a diabetic rat model.

OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Validation and assessment of an antibiotic-based, aseptic decontamination manufacturing protocol for therapeutic, vacuum-dried human amniotic membrane.

Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and -negative bacteria.

Readiness to Change Over Time: Change Commitment and Change Efficacy in a Workplace Health-Promotion Trial.

INTRODUCTION: Organizational readiness to change may be a key determinant of implementation success and a mediator of the effectiveness of implementation interventions. If organizational readiness can be reliably and validly assessed at the outset of a change initiative, it could be used to assess the effectiveness of implementation-support activities by measuring changes in readiness factors over time. METHODS: We analyzed two waves of readiness-to-change survey data collected as part of a three-arm, randomized controlled trial to implement evidence-based health promotion practices in small worksites in low-wage industries. We measured five readiness factors: context (favorable broader conditions); change valence (valuing health promotion); information assessment (demands and resources to implement health promotion); change commitment (an intention to implement health promotion); and change efficacy (a belief in shared ability to implement health promotion). We expected commitment and efficacy to increase at intervention sites along with their self-reported effort to implement health promotion practices, termed wellness-program effort. We compared means between baseline and 15 months, and between intervention and control sites. We used linear regression to test whether intervention and control sites differed in their change-readiness scores over time. RESULTS: Only context and change commitment met reliability thresholds. Change commitment declined significantly for both control (-0.39) and interventions sites (-0.29) from baseline to 15 months, while context did not change for either. Only wellness program effort at 15 months, but not at baseline, differed significantly between control and intervention sites (1.20 controls, 2.02 intervention). Regression analyses resulted in two significant differences between intervention and control sites in changes from baseline to 15 months: (1) intervention sites exhibited significantly smaller change in context scores relative to control sites over time and (2) intervention sites exhibited significantly higher changes in wellness program effort relative to control sites. DISCUSSION: Contrary to our hypothesis, change commitment declined significantly at both Healthlinks and control sites, even as wellness-program effort increased significantly at HealthLinks sites. Regression to the mean may explain the decline in change commitment. Future research needs to assess whether baseline commitment is an independent predictor of wellness-program effort or an effect modifier of the HealthLinks intervention.

Development and Pilot Test of the Workplace Readiness Questionnaire, a Theory-Based Instrument to Measure Small Workplaces' Readiness to Implement Wellness Programs.

PURPOSE: To develop a theory-based questionnaire to assess readiness for change in small workplaces adopting wellness programs. DESIGN: In developing our scale, we first tested items via "think-aloud" interviews. We tested the revised items in a cross-sectional quantitative telephone survey. SETTING: The study setting comprised small workplaces (20-250 employees) in low-wage industries. SUBJECTS: Decision-makers representing small workplaces in King County, Washington (think-aloud interviews, n = 9), and the United States (telephone survey, n = 201) served as study subjects. MEASURES: We generated items for each construct in Weiner's theory of organizational readiness for change. We also measured workplace characteristics and current implementation of workplace wellness programs. ANALYSIS: We assessed reliability by coefficient alpha for each of the readiness questionnaire subscales. We tested the association of all subscales with employers' current implementation of wellness policies, programs, and communications, and conducted a path analysis to test the associations in the theory of organizational readiness to change. RESULTS: Each of the readiness subscales exhibited acceptable internal reliability (coefficient alpha range, .75-.88) and was positively associated with wellness program implementation ( p < .05). The path analysis was consistent with the theory of organizational readiness to change, except change efficacy did not predict change-related effort. CONCLUSION: We developed a new questionnaire to assess small workplaces' readiness to adopt and implement evidence-based wellness programs. Our findings also provide empirical validation of Weiner's theory of readiness for change.

HealthLinks randomized controlled trial: Design and baseline results.

Small employers, especially those in low-wage industries, frequently lack the capacity and resources to implement evidence-based health promotion interventions without support and assistance. The purpose of this paper is to (a) describe the intervention design and study protocol of the HealthLinks Trial and (b) report baseline findings. This study is a three-arm randomized controlled trial testing the impact of the HealthLinks intervention on worksites' adoption and implementation of evidence-based interventions. Group 1 will receive HealthLinks, Group 2 will receive HealthLinks plus wellness committees, and Group 3 will be a delayed control group. Seventy-eight employers are participating in the study; and 3302 employees across the worksites participated in the baseline data collection. Employers and employees will participate in follow-up surveys at one and two years after baseline to measure implementation (one year) and maintenance (two years) of HealthLinks interventions. Study outcomes will determine whether HealthLinks is an effective approach to increasing evidence-based health promotion in small, low-wage worksites and whether wellness committees are a capacity-building tool that increases HealthLinks' effectiveness.

Promoting Influenza Vaccination to Restaurant Employees.

PURPOSE: To evaluate an evidence-based workplace approach to increasing adult influenza vaccination levels applied in the restaurant setting DESIGN: We implemented an intervention and conducted a pre/post analysis to determine effect on vaccination. SETTING: Eleven Seattle-area restaurants. SUBJECTS: Restaurants with 25+ employees speaking English or Spanish and over 18 years. INTERVENTION: Restaurants received influenza vaccination promotion materials, assistance arranging on-site vaccination events, and free influenza vaccinations for employees. MEASURES: Pre/post employee surveys of vaccination status with direct observation and employer interviews to evaluate implementation. ANALYSIS: We conducted descriptive analysis of employee survey data and performed qualitative analysis of implementation data. To assess intervention effect, we used a mixed-effects logistic regression model with a restaurant-specific random effect. RESULTS: Vaccination levels increased from 26% to 46% (adjusted odds ratio 2.33, 95% confidence interval 1.69, 3.22), with 428 employees surveyed preintervention, 305 surveyed postintervention, and response rates of 73% and 55%, respectively. The intervention was effective across subgroups, but there were restaurant-level differences. CONCLUSION: An access-based workplace intervention can increase influenza vaccination levels in restaurant employees, but restaurant-level factors may influence success.

Feasibility of Workplace Health Promotion for Restaurant Workers, Seattle, 2012.

INTRODUCTION: Restaurant workers are a large population at high risk for tobacco use, physical inactivity, and influenza. They are difficult to reach with health care interventions and may be more accessible through workplaces, yet few studies have explored the feasibility of workplace health promotion in this population. This study sought to identify barriers and facilitators to promotion of tobacco cessation, physical activity, and influenza vaccination in restaurants. METHODS: Moderators conducted 7 focus groups, 3 with restaurant owners and managers, 2 with English-speaking workers, and 2 with Spanish-speaking workers. All groups were recorded, and recordings were transcribed and uploaded to qualitative-analysis software. Two researchers coded each transcript independently and analyzed codes and quotations for common themes. RESULTS: Seventy people from the restaurant industry participated. Barriers to workplace health promotion included smoking-break customs, little interest in physical activity outside of work, and misinformation about influenza vaccinations. Facilitators included creating and enforcing equitable break policies and offering free, on-site influenza vaccinations. Spanish-speakers were particularly amenable to vaccination, despite their perceptions of low levels of management support for health promotion overall. Owners required a strong business case to consider investing in long-term prevention for their employees. CONCLUSION: Tobacco cessation and influenza vaccinations are opportunities for health promotion among restaurant workers, whereas physical activity interventions face greater challenges. Promotion of equitable breaks, limited smoking-break policies, and free, on-site influenza vaccinations could improve health for restaurant workers, who often do not have health insurance. Workplace interventions may be particularly important for Hispanic workers who have additional access barriers.

Process evaluation of a regional public health model to reduce chronic disease through policy and systems changes, Washington State, 2010-2014.

INTRODUCTION: Although the regionalization of public health systems has been well documented in the case of emergency preparedness, there is little literature on the application of regional approaches to other aspects of public health. From 2011 through 2014 the Washington State Department of Health implemented a Community Transformation Grant to support community-level policy and systems changes to decrease chronic disease risk factors and increase access to clinical preventive services. The Department of Health implemented the grant through a regional model, grouping 32 of the state's 35 local health jurisdictions into 5 regions. Our process evaluation identifies the challenges and facilitators to Community Transformation Grant planning and implementation. METHODS: We conducted 34 key informant interviews with people directly involved in the implementation of the Community Transformation Grant. We interviewed state and local partners, including representatives from each region, the Department of Health, external consultants, and regional partners. We collected data from October 2013 through July 2014. RESULTS: Challenges for planning, building, and implementing a regional model for chronic disease prevention included stakeholder buy-in, regional geography, and communication; facilitators included shared regional history and infrastructure, strong leadership, collaborative relationships, shared vision and goals, sufficient funding, and direct technical assistance and training. CONCLUSION: Lessons learned in Washington State provide a foundation for other states interested in using a regional approach to reduce chronic disease risk. Policy and systems changes require adequate time, funding, and staffing. States and funders should work closely with local leaders to address these challenges and facilitators.

Influenza vaccination status and attitudes among restaurant employees.

CONTEXT: Restaurant employees represent a substantial portion of the US workforce, interact closely with the public, and are at risk for contracting influenza, yet their influenza vaccination rates and attitudes are unknown. OBJECTIVE: Assess influenza vaccination rates and attitudes among Seattle restaurant employees, to identify factors that could enhance the success of a restaurant-based vaccination program. DESIGN: In 2012, we invited employees of Seattle restaurants to complete an anonymous paper survey assessing participant demographics, previous influenza vaccination status, and personal attitudes toward influenza vaccination (using a 5-point scale). SETTING: Sit-down, full service restaurants in or near Seattle, Washington, were eligible if they had no previous history of offering worksite influenza vaccinations and had more than 20 employees who were older than 18 years and spoke either English or Spanish. PARTICIPANTS: We invited staff in all restaurant positions (servers, bussers, kitchen staff, chefs, managers, etc) to complete the survey, which was available in English and Spanish. RESULTS: Of 428 restaurant employees surveyed, 26% reported receiving the seasonal influenza vaccine in 2011-2012 (response rate = 74%). Across 8 attitude statements, participants were most likely to agree that the vaccine is not too expensive (89%), and least likely to agree that it is relevant for their age group (25%), or normative at their workplace (13%). Vaccinated participants reported significantly more positive attitudes than unvaccinated participants, and Hispanics reported significantly more positive attitudes than non-Hispanic whites. CONCLUSIONS: Increasing influenza vaccination rates among restaurant employees could protect a substantial portion of the US workforce, and the public, from influenza. Seattle restaurant employees have low vaccination rates against seasonal influenza. Interventions aimed at increasing vaccination among restaurant employees should highlight the vaccine's relevance and effectiveness for working-age adults.

Opportunities for improving cancer prevention at federally qualified health centers.

As the Affordable Care Act unfolds, federally qualified health centers (FQHCs) will likely experience an influx of newly insured, low-income patients at disparate risk for cancer. Cancer-focused organizations are seeking to collaborate with FQHCs and the Primary Care Associations (PCAs) that serve them, to prevent cancer and reduce disparities. To guide this collaboration, we conducted 21 interviews with representatives from PCAs and FQHCs across four western states. We asked about: FQHC priorities, barriers and facilitators to cancer prevention, the PCA-FQHC relationship, and collaboration opportunities for external organizations. FQHC priorities include medical home transformation, electronic health records, and clinical care; prevention efforts must integrate with these. Barriers to cancer prevention include competing priorities, inadequate patient insurance, and lack of reimbursement, while facilitators are the presence of patient navigators and cancer-related performance measures. Collaboration opportunities for external organizations include dissemination of culturally appropriate educational materials and support for patient navigators.

Augmented dried versus cryopreserved amniotic membrane as an ocular surface dressing.

PURPOSE: Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material. METHODS: AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays. RESULTS: Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM. CONCLUSIONS: Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.

Risk factors for ischaemic stroke.

Stroke is a leading cause of morbidity and mortality in western populations, with up to 40% of survivors not expected to recover independence from severe disabilities. This equates to an immense financial burden on health systems worldwide. Hence further education is required to inform individuals of the risks to promote secondary prevention strategies in future generations. Stroke is a heterogeneous, multifactorial disease regulated by modifiable and nonmodifiable risk factors. Modifiable factors include a history of high blood pressure, diabetes mellitus and coronary heart disease. Nonmodifiable factors include age, sex and race. Other less-well documented risk factors include geographic location, socioeconomic status and alcoholism. Approximately 80% of stroke events could be reduced by making simple lifestyle modifications. Further studies are required to clarify the role and interplay of the risk factors outlined to give a more comprehensive understanding of stroke and to aid and drive the development of more effective stroke prevention programs, in high risk groups.

Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells.

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remains to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [(3)H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50 microM) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 microM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction pathways.